It is known that certain peptides can be used as highly specific vehicles for the delivery of radioisotopes to target organs, tumors or thrombi in vivo. For example, somatostatin receptor-positive human tumors can be detected using radioiodinated analogues of somatostatin (Lamberts, S. W. J. et al., N. Eng. J. Med. (1990) 323: 1246-1249; Krenning E. P. et al., Lancet (1989) 242-245; Bakker W. H. et al., J. Nucl. Med. (1990) 32: 1501-1509). Rodwell et al., U.S. Pat. No. 5,196,510, showed that a radiolabeled peptide containing the amino acid sequence, RGD, could localize to thrombi in vivo, whereby thrombi could be scintigraphically imaged.
In nuclear medicine, the radiometal technetium-99m (Tc-99m) is a preferred isotope for scintigraphic imaging applications (Pinkerton et al., J. Chem. Educ. (1985) 62: 965). Technetium is one of a class of metals ions which forms strong coordination bonds with sulfur-containing compounds, particularly thiols (e.g., metallothionein), but also with thioureas (Kopunec et al., Radiochem. Radioanal. Lett. (1977) 29: 171).
Methods for the direct labeling of peptides with technetium have been reported which require the partial reduction of protein disulfide linkages to generate the free thiol groups that are capable of binding radiometals, specifically Tc-99m. Dean, U.S. Pat. No. 5,225,180, describes derivatives of somatostatin which contain at least 2 cysteine residues. The cysteine residues typically form a disulfide bond which, on reduction, form two sulfhydryl groups that are capable of coordinating to Tc-99m. Similarly, cysteine-containing amino acid sequences, which are derived from metallothionein, bind metals through these sulfhydryl moieties and can be incorporated into the amino acid sequence of targeting peptides to make radiopharmaceuticals (Rodwell, supra; Shoemaker, International Patent Publication No. WO90/06323). Alternatively, metallothionein or fragments thereof can be used to label proteins indirectly by conjugating them to biologically active molecules as described by Tolman, U.S. Pat. No. 4,732,864.
Direct radiolabeling methods, while advantageous, may not be possible or desirable in some cases. Many peptides, especially small ones, do not contain disulfide moieties and, therefore, cannot be labeled via direct methods without further chemical or recombinant modification of the peptide or protein to include disulfide bonds. Moreover, the reduction, itself, of the disulfide bonds can denature, fragment or aggregate the peptide or cause deviations from native conformations that may compromise peptide-receptor binding and, otherwise, compromise the biological targeting capability of the radiolabeled product. If the targeting protein contains more than one disulfide, the current methods provide no way of distinguishing between the disulfides and, hence, no means for directing the metal to a particular metal-binding site.
It has further been shown that targeting proteins can also be radiolabeled by covalently linking or conjugating chelating ligands capable of binding metals to the targeting proteins. Albert et al., International Patent Publication No. WO91/01144, disclose biologically active peptides bearing at least one chelating ligand linked to an amino group of the peptide useful as a radiopharmaceutical for in vivo imaging of target tissues or for therapy. The chelating ligand must be linked to an amino group that is not involved in the binding of the peptide to the targeted receptor. The chelating ligands disclosed include polyamine or imine carboxylic acid chelators, e.g., EDTA, DTPA, etc.; C-functionalized tetraazacyclododecanetetraacetic acids; N-substituted or C-substituted macrocyclic amines; bis-aminothiol alkylene derivatives; ethylene dithiosemicarbazone derivatives; ligands derived from propylene amine oxime derivatives; and ligands derived from diamide dimercaptides. Fritzberg, European Patent No. 0 188 256 A2, describes N,N'-bismercaptoacetyl-w,(w-x)-diamino carboxylic acid esters conjugated to polypeptides.
Yokoyama et al., U.S. Pat. No. 4,287,362; Int. J. Nucl. Med. Biol. (1986) 12: 425; J. Nucl. Med. (1987) 28: 1027, describe bifunctional chelators similar to Albert et al., supra, based on N-methylthiosemicarbazone derivatives of 1,2-dicarbonyl compounds. These bifunctional compounds have two N-methylthiosemicarbazone binding groups on one side of the chelating ligand while the opposite side contains a functional group to which the targeting protein is covalently attached. Analogous systems are described by Wu, EP 0306168, which are also based on thiosemicarbazone derivatives of dicarbonyl compounds. Arano et al., Int. J. Nucl. Med. Biol. (1986) 12 (6): 425-430, teach the use of a chelating agent, p-carboxyethyl-phenylglyoxal-di(N-methylthiosemicarbazone), to radiolabel biologically interesting molecules.
Troutner, U.S. Pat. No. 5,101,041, describes functionalized triamine chelating ligands that are covalently attached to proteins. McBride et al., U.S. Pat. No. 5,080,884, disclose hydrocarbylphenyl diaminodithiol radionuclide complexes. No biological targeting moieties are disclosed.
Thus, previous work has relied on the covalent attachment of a chelating ligand to a biological targeting moiety, like a protein, to provide a targettable radiolabel. Unfortunately, chemistries for conjugating chelators to targeting peptides are often not compatible with the biological targeting moiety. Hence, the biological activity of the targeting peptides or proteins is not always preserved. Additionally, free thiols are somewhat unstable to oxidation/dimerization and may, in turn, reduce disulfide bonds or react with a free sulfhydryl group elsewhere in the same peptide or in neighboring peptides. Free cysteines may recombine to disulfide groups through air oxidation, eliminating metal coordinating thiol groups. The thiol functions in typical chelators that can be attached to peptides must be protected during final steps of synthesis and then deprotected just before technetium binding. These steps are somewhat cumbersome for radiolabeling. Such chemistries can also be difficult or exceedingly tedious, adding numerous steps (including additional purification procedures) and expense to the overall synthesis of the targeting-chelator compounds. Furthermore, such manipulations can unduly complicate the radiolabeling procedure. Moreover, the covalent linkage provided by current conjugation chemistries is not always stable. For example, thiosemicarbazone derivatives or imine derivatives, which are formed by a condensation reaction that results in the removal of water, may be subsequently susceptible to undesired hydrolysis. Such compounds can be expected to hydrolyze easily in an acidic environment or in vivo prematurely, leading to the disintegration of the targeting protein metal-chelate conjugate.
Hence, there remains a need for a simple targettable construct that possesses strong label-binding characteristics and in vivo or in vitro targeting specificity. A desirable targettable construct would be one that is easily made, with a minimum number of synthetic steps, yet retain the versatility to accommodate a number of types of labels and starting biological targeting species, particularly proteins and peptides. Such a desirable targettable construct would even be directable to more than one type of receptor at any given time. In addition, such a desirable targettable label would be amenable to the on-site radiolabeling procedure that is typical of short half-life radiopharmaceutical products. While being especially useful in in vivo or in vitro diagnostic applications, such a targettable construct would also find application in the therapeutics arena.